Infrared spectroscopy of synovial fluid as a potential screening approach for the diagnosis of naturally occurring canine osteoarthritis associated with cranial cruciate ligament rupture.

Objective: To evaluate infrared (IR) spectroscopy of synovial fluid (SF) as tool to differentiate between knees of dogs with naturally occurring OA associated with cranial cruciate ligament rupture (CrCLR) and controls. Method: 104 adult dogs with CrCLR (affected group) and 50 adult control dogs were recruited in a prospective observational study. Synovial fluid (SF) samples were collected preoperatively from dogs with CrCLR and from a subset of these at 4-, and 12-week post-surgery. Knee samples were collected bilaterally once from control dogs. Dried synovial fluid films were made, and IR absorbance spectra acquired. After preprocessing, partial least squares discriminant analysis (PLS-DA) and ANOVA-simultaneous component analysis (ASCA) were used to evaluate group and temporal differences, and to develop predictive models. Results: There were statistically significant spectral differences between the SF of OA affected and control dogs at all three time-points (P < 0.001). Pairwise comparison of spectral SF of knees with CrCLR over time showed statistically significant differences amongst all three time-points (P < 0.001). The predictive model for identifying the affected group from control had sensitivity, specificity and overall accuracy of 97.6%, 99.7% and 98.6%, respectively. Conclusions: The findings demonstrate the ability of FTIR-spectroscopy of synovial fluid combined with chemometric methods to accurately differentiate dogs with OA secondary to CrCLR from controls. The role of this IR-based screening test as a diagnostic and monitoring biomarker for OA specific to the joint being sampled warrants further investigation.

In vitro cultivation of Hematodinium sp. isolated from Atlantic snow crab, Chionoecetes opilio: partial characterization of late developmental stages.

Hematodinium is a parasitic dinoflagellate of numerous crustacean species, including the economically important Atlantic snow crab, Chionoecetes opilio. The parasite was cultured in vitro in modified Nephrops medium at 0 °C and a partial characterization of the life stages was accomplished using light and transmission electron microscopy (TEM). In haemolymph from heavily infected snow crabs two life stages were detected; amoeboid trophonts and sporonts. During in vitro cultivation, several Hematodinium sp. life stages were observed: trophonts, clump colonies, sporonts, arachnoid sporonts, sporoblasts and dinospores. Cultures initiated with sporonts progressed to motile dinospores; however, those initiated with amoeboid trophonts proliferated, but did not progress or formed schizont-like stages which were senescent artefacts. Plasmodial stages were associated with both trophonts and sporonts and could be differentiated by the presence of trichocysts on TEM. Macrodinospores were observed but not microdinospores; likely due to the low number of Hematodinium sp. cultures that progressed to the dinospore stage. No early life stages including motile filamentous trophonts or gorgonlocks were observed as previously noted in Hematodinium spp. from other crustacean hosts. All Hematodinium sp. life stages contained autofluorescent, membrane-bound electron dense granules that appeared to degranulate or be expelled from the cell during in vitro cultivation.

The effects of temperature and body size on immunological development and responsiveness in juvenile shortnose sturgeon (Acipenser brevirostrum).

Sturgeon are an important evolutionary taxa of which little is known regarding their responses to environmental factors. Water temperature strongly influences growth in fish; however, its effect on sturgeon immune responses is unknown. The objective of this study was to assess how 2 different temperatures affect immune responses in shortnose sturgeon (Acipenser brevirostrum) relevant immune organs such as the meningeal myeloid tissue, spleen, thymus and skin. These responses were studied in 2 different sizes of same age juvenile sturgeon kept at either 11 °C or 20 °C (4 treatment groups), before and after exposure to an ectoparasitic copepod (Dichelesthium oblongum). Based on a differential cell count, temperature was found to strongly influence immune cell production in the meningeal myeloid tissue, regardless of the fish sizes considered. Morphometric analysis of splenic white pulp showed a transient response to temperature. There were no differences between the groups in the morphometric analysis of thymus size. Splenic IRF-1 and IRF-2 had similar expression profiles, significantly higher in fish kept at 20 °C for the first 6 weeks of the study but not by 14 weeks. In the skin, IRF-1 was significantly higher in the fish kept at 11 °C over the first 6 weeks of the study. IRF-2 had a similar profile but there were no differences between the groups by the end of the trial. In conclusion, higher water temperatures (up to 20 °C) may have beneficial effects in maximizing growth and improving immunological capacity, regardless of the fish sizes considered in this study.

Ontogeny of the immune system in Acipenserid juveniles.

Sturgeon aquaculture has increased considerably worldwide but little is known about their immunological development and competence in early life stages. Culture of larvae is one of the most critical stages in intensive sturgeon farming, often associated with high mortality rates. The objective of this study was to characterize the developmental morphology (light and transmission electron microscopy, LM and TEM) of the meningeal myeloid tissue, spleen and thymus in Atlantic sturgeon (Acipenser oxyrinchus oxyrinchus) from hatching until 5 months old (2895°C·day (dd)). The spleen was first visible on 541 dd larvae LM sections and the other two immune organs in 768 dd samples (approximately 400 and 600 dd after onset of feeding). Generally, younger fish had significantly higher percentages of undifferentiated cells (meningeal myeloid tissue and spleen) and effective adaptive immune competence would not be expected in these fish on the onset of feeding, but further functional immune assessment is needed.

Estimation of total glomerular number using an integrated disector method in embryonic and postnatal kidneys.

Congenital Anomalies of the Kidney and Urinary Tract (CAKUT) are a polymorphic group of clinical disorders comprising the major cause of renal failure in children. Included within CAKUT is a wide spectrum of developmental malformations ranging from renal agenesis, renal hypoplasia and renal dysplasia (maldifferentiation of renal tissue), each characterized by varying deficits in nephron number. First presented in the Brenner Hypothesis, low congenital nephron endowment is becoming recognized as an antecedent cause of adult-onset hypertension, a leading cause of coronary heart disease, stroke, and renal failure in North America. Genetic mouse models of impaired nephrogenesis and nephron endowment provide a critical framework for understanding the origins of human kidney disease. Current methods to quantitate nephron number include (i) acid maceration (ii) estimation of nephron number from a small number of tissue sections (iii) imaging modalities such as MRI and (iv) the gold standard physical disector/fractionator method. Despite its accuracy, the physical disector/fractionator method is rarely employed because it is labour-intensive, time-consuming and costly to perform. Consequently, less rigourous methods of nephron estimation are routinely employed by many laboratories. Here we present an updated, digitized version of the physical disector/fractionator method using free open source Fiji software, which we have termed the integrated disector method. This updated version of the gold standard modality accurately, rapidly and cost-effectively quantitates nephron number in embryonic and post-natal mouse kidneys, and can be easily adapted for stereological measurements in other organ systems.

Les anomalies congénitales du rein et des voies urinaires (Congenital Anomalies of the Kidney and Urinary Tract, CAKUT) désignent un groupe polymorphe d'entités cliniques qui constitue la cause la plus fréquente d'insuffisance rénale chez l'enfant. Le CAKUT comprend aussi un grand nombre de malformations développementales, dont le syndrome de Potter, l'hypoplasie rénale, ainsi que la dysplasie rénale (maldifférentiation des tissus rénaux), toutes caractérisées par un déficit de néphrons. On reconnaît de plus en plus une masse néphronique congénitale réduite, d'abord présentée dans l'hypothèse de Brenner, comme une cause de l'hypertension chez l'adulte, de coronaropathie, d'AVC, et d'insuffisance rénale en Amérique du Nord. Les modèles génétiques de souris comportant une détérioration de la fonction rénale et de la masse néphronique fournissent un cadre pour permettre la compréhension de l'origine des néphropathies chez l'humain. Les méthodes actuelles de quantification des néphrons comprennent (i) la macération acide (ii) l'estimation du nombre de néphrons à partir d'une petite quantité de tissus sectionnés (iii) les modes d'imagerie tels que l'IRM et (iv) la technique de référence du disecteur et fractionnement. Malgré sa précision, cette dernière méthode n'est employée que rarement, puisqu'elle requiert main-d'œuvre, temps et argent. Par conséquent, plusieurs laboratoires emploient systématiquement des méthodes moins rigoureuses d'estimation du nombre de néphrons. Nous présentons ici une version mise à jour et numérisée de la technique du disecteur et fractionnement, que nous appelons la technique intégrée du disecteur, en utilisant Fiji, un logiciel ouvert et gratuit. Cette version mise à jour de la modalité de référence permet de quantifier les néphrons de manière précise, rapide et rentable dans les reins de souris à l'état embryonnaire ou postnatal, et peut aisément être adaptée aux mesures stéréologiques d'autres organes.

Osteoprogenitor cell therapy in an equine fracture model.

OBJECTIVE: To compare the efficacy of osteoprogenitors in fibrin glue to fibrin glue alone in bone healing of surgically induced ostectomies of the fourth metacarpal bones in an equine model. STUDY DESIGN: Experimental. ANIMALS: Adult horses (n = 10). METHODS: Segmental ostectomies of the 4th metacarpal bone (MC4) were performed bilaterally in 10 horses. There was 1 treatment and 1 control limb in each horse. Bone defects were randomly injected with either fibrin glue and osteoprogenitor cells or fibrin glue alone. Radiography was performed every week until the study endpoint at 12 weeks. After euthanasia, bone healing was evaluated using radiography and histology. Analysis of radiographic data was conducted using a linear-mixed model. Analysis of histologic data was conducted using a general linear model. Statistical significance was set at P < .05. RESULTS: Radiographic grayscale data as a measure of bone healing revealed no significant difference between treatment and control limbs. Radiographic scoring results also showed that the treatment effect was not significant. Histologic analysis was consistent with radiographic analysis showing no significant difference between the area of bone present in treatment and control limbs. CONCLUSION: Injection of periosteal-derived osteoprogenitors in a fibrin glue carrier into surgically created ostectomies of MC4 does not accelerate bone healing when compared with fibrin glue alone.

Ontogeny and disease responses of Langerhans-like cells in lymphoid tissues of salmonid fish.

The ontogeny and disease responses of Langerhans-like cells within lymphoid tissues of Atlantic salmon, Salmo salar, and rainbow trout, Oncorhynchus mykiss, were investigated. These cells were studied in situ with the use of two markers: the ultrastructural presence of Birbeck-like granules and immunohistochemistry with an antibody against human langerin/CD207 that cross-reacts with salmonid tissues. The appearance of Birbeck-like granules was observed in rainbow trout at 2 weeks post-hatch (PH) in the thymus and anterior kidney prior to the development of the spleen. Spleen first appeared at 3 weeks PH in both Atlantic salmon and rainbow trout, and Birbeck-like granules were observed within cells of the newly developed spleens. The cross-reactivity of langerin as seen by immunohistochemistry was not clearly observed in kidney and spleen until 9 weeks PH, when a strong cytoplasmic reaction was observed. To study langerin-positive cells in spleen and kidney during disease, microsporidial gill disease (MGD) in rainbow trout was used as a known disease model inducing a strong cell-mediated adaptive immune response. Langerin-positive cells in healthy fish were seen predominantly in the spleen, and only low numbers were present in the anterior kidney. During MGD, langerin-positive cell numbers were elevated in the anterior kidney and were significantly higher during 5, 6, and 10 weeks post-exposure (PE) compared with healthy control tissue. During MGD, the distribution of langerin-positive cells in the spleen and anterior kidney shifted from having significantly higher numbers of cells in the spleen than in the kidney in controls and at 1 and 4 weeks PE to having a similar distribution of the cells in the two organs at 2, 3, 5, and 6 weeks PE. By 10 weeks PE, significantly higher numbers of langerin-positive cells occurred in the anterior kidney compared with the spleen.

Comparative ultrastructure of Langerhans-like cells in spleens of ray-finned fishes (Actinopterygii).

We studied the morphology and occurrence of splenic Langerhans-like (LL) cells in species representing 11 orders of ray-finned fishes, Actinopterygii. LL cells were frequent in spleen tissue of species among Cypriniformes, Esociformes, Salmoniformes, and Pleuronectiformes. These cells contained granules which resembled Birbeck granules known to occur in mammalian Langerhans cells. The ultrastructure of LL cells in Northern pike, Esox lucius, and in Atlantic halibut, Hippoglossus hippoglossus were similar to those reported in salmonids. LL cells found in cyprinids shared some characteristics with the LL cells in other Actinopterygii species, although unique structures distinguished them from the latter. They contained dense bodies within the Birbeck-like (BL) granules, a characteristic that was never observed in species outside the Cypriniformes. Two types of BL granules were characterized in cyprinid LL cells. The ultrastructure of BL granules across the species is discussed. LL cells in all Actinopterygii species demonstrated close contacts with nearby cells, characterized by adherens-like junctions. Additionally, multivesicular bodies were present within the cytoplasm and large aggregates of exosomes were observed closely associated with the plasma membrane suggesting their release from the cells. These structures are discussed in relation to mammalian dendritic cells. Macrophages found in European perch, Perca fluviatilis, blue gourami, Trichogaster trichopterus, and Atlantic halibut, Hippoglossus hippoglossus contained lysosomes and residual bodies with structures resembling Birbeck granules. These granules and cells were clearly distinct from LL cells.

Developmental anatomy of lampreys.

Lampreys are a group of aquatic chordates whose relationships to hagfishes and jawed vertebrates are still debated. Lamprey embryology is of interest to evolutionary biologists because it may shed light on vertebrate origins. For this and other reasons, lamprey embryology has been extensively researched by biologists from a range of disciplines. However, many of the key studies of lamprey comparative embryology are relatively inaccessible to the modern scientist. Therefore, in view of the current resurgence of interest in lamprey evolution and development, we present here a review of lamprey developmental anatomy. We identify several features of early organogenesis, including the origin of the nephric duct, that need to be re-examined with modern techniques. The homologies of several structures are also unclear, including the intriguing subendothelial pads in the heart. We hope that this review will form the basis for future studies into the phylogenetic embryology of this interesting group of animals.

Langerin/CD207 positive dendritic-like cells in the haemopoietic tissues of salmonids.

The presence of dendritic cells in fish is studied with immunohistochemistry using a commercially available antibody developed against Langerin/CD207 present in human Langerhans cells. Langerin/CD207, a protein known to be associated with the development of Birbeck granules in human and murine systems, was found to be expressed within the cytoplasm of spleen and head kidney cells of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss). Reactivity was also observed within a few number of cells within the head kidney of Atlantic salmon, but not observed in any other tissues examined. Immunohistochemical results showed Langerin/CD207 reactivity in the cytoplasm of cells in Atlantic salmon and rainbow trout comparable to reactivity seen in human Langerhans cells. The results in this study further corroborate the presence of dendritic cells with remarkable similarities to human Langerhans cells in the spleens and to a lesser extent in head kidney of salmonids.

Infectious salmon anaemia virus replication and induction of alpha interferon in Atlantic salmon erythrocytes.

BACKGROUND: Infectious salmon anaemia (ISA) virus (ISAV), which causes ISA in marine-farmed Atlantic salmon, is an orthomyxovirus belonging to the genus Isavirus, family Orthomyxoviridae. ISAV agglutinates erythrocytes of several fish species and it is generally accepted that the ISAV receptor destroying enzyme dissolves this haemagglutination except for Atlantic salmon erythrocytes. Recent work indicates that ISAV isolates that are able to elute from Atlantic salmon erythrocytes cause low mortality in challenge experiments using Atlantic salmon. Previous work on ISAV-induced haemagglutination using the highly pathogenic ISAV strain NBISA01 and the low pathogenic ISAV strain RPC/NB-04-0851, showed endocytosis of NBISA01 but not RPC/NB-04-0851. Real-time RT-PCR was used to assess the viral RNA levels in the ISAV-induced haemagglutination reaction samples, and we observed a slight increase in viral RNA transcripts by 36 hours in the haemagglutination reaction with NBISA01 virus when the experiment was terminated. However, a longer sampling interval was considered necessary to confirm ISAV replication in fish erythrocytes and to determine if the infected cells mounted any innate immune response. This study examined the possible ISAV replication and Type I interferon (IFN) system gene induction in Atlantic salmon erythrocytes following ISAV haemagglutination. RESULTS: Haemagglutination assays were performed using Atlantic salmon erythrocytes and one haemagglutination unit of the two ISAV strains, NBISA01 and RPC/NB-04-0851, of differing genotypes and pathogenicities. Haemagglutination induced by the highly pathogenic NBISA01 but not the low pathogenic RPC/NB-04-0851 resulted in productive infection as evidenced by increased ISAV segment 8 transcripts and increase in the median tissue culture infectious dose (TCID50) by 5 days of incubation. Moreover, reverse transcription (RT) quantitative PCR used to compare mRNA levels of key Type I IFN system genes in erythrocyte lysates of haemagglutination reactions with the two ISAV strains showed a higher relative fold increase of IFN-alpha in NBISA01 haemagglutinations compared to RPC/NB-04-085-1 haemagglutinations (33.0 - 44.26 relative fold increase compared to 11.29). Erythrocytes exposed to heat-inactivated virus or to polyinosinic:polycytidylic acid (polyI:C) or to L-15 medium alone (negative control assays) had minimal late induction (<3.5 relative fold increase) of STAT1 and/or ISG15 and Mx genes, whereas erythrocytes exposed to UV-inactivated virus lacked any cytokine induction. CONCLUSION: ISAV-induced haemagglutination by a highly pathogenic virus strain results in virus uptake and productive infection of Atlantic salmon erythrocytes accompanied by significant induction of IFN-alpha. This study also highlights the critical role of ISAV strain variation in the initial stages of the virus-cell interaction during haemagglutination, and possibly in the pathogenesis of ISA. Moreover, the study shows for the first time that fish erythrocytes immunologically respond to ISAV infection.

Effects of dexamethasone on host innate and adaptive immune responses and parasite development in rainbow trout Oncorhynchus mykiss infected with Loma salmonae.

The effects of dexamethasone (dex) treatment on infections with the microsporidian parasite, Loma salmonae and the effects of dex on initiation of the adaptive immune response were investigated in rainbow trout, Oncorhynchus mykiss experimentally infected with the parasite. Dex treatment resulted in significantly higher infections with the parasite in the gills and other internal organs, suggesting that dex inhibits aspects of the innate immune response to L. salmonae; the heavier infections in the gills and organs of rainbow trout resembled infections seen in Chinook salmon. Mean xenoma counts per microscope field in the gills of fish infected with L. salmonae treated with dex or left untreated were 169 and 30, respectively. Although higher numbers of xenomas were observed in dex treated fish, the xenomas were generally smaller in size than in infected control fish. The xenomas in dex treated fish showed morphological signs of degeneration including loss and degeneration of early parasite stages, accumulation of amorphous material in xenomas, and infiltration with phagocytic cells containing degenerated parasites. The xenomas in infected untreated fish had larger xenomas with a more uniform size and contained identifiable parasite stages in the cytoplasm. According to this study, once fish have developed an adaptive immune response to the parasite by previous exposure, then fish have 100% protection to reinfection even when treated with heavy doses of dex. L. salmonae immune fish treated or untreated with dex during reinfection with the parasite developed no xenomas in the gills 6 weeks post reinfection. These results indicate that once the cellular response is primed to L. salmonae, then dex related immunosuppression does not reduce the effectiveness of the adaptive immune response.

Mutated ATP synthase induces oxidative stress and impaired insulin secretion in beta-cells of female BHE/cdb rats.

BACKGROUND: Adenosine triphosphate (ATP) is a critical determinant of beta-cell insulin secretion in response to glucose. BHE/cdb rats have a mutation in ATP synthase that limits ATP production, yet develop mild diabetes only with ageing. We investigated the cellular basis for reduced insulin secretion and compensatory mechanisms that mitigate the effects of the ATP synthase mutation. METHODS: In vitro beta-cell function in isolated islets and expression of key regulatory genes was compared with in vivo oral glucose tolerance and insulin sensitivity in BHE/cdb and control rats. RESULTS: BHE/cdb rat islets had reduced responsiveness to glucose stimulation and ATP content was 35% lower than in control islets. Oral glucose tolerance was impaired at both 21 and 43 weeks of age because of a reduction in glucose-stimulated insulin secretion (GSIS). An increase in inducible nitric oxide synthase (INOS, 3-fold) and manganese superoxide dismutase (MnSOD, 1.6-fold), detection of nitrotyrosine, beta-cell apoptosis, and nucleocytoplasmic translocation of pancreas duodenum homeobox-1 (PDX-1) in beta-cells indicated increased oxygen radical formation. However, BHE/cdb rats partially compensated for low glucose responsiveness by increasing the number of small islets and beta-cell hypertrophy. There was also an increase in the proportion of mature insulin relative to proinsulin (PI) detected within beta-cell granules. Increased activation of AMP-dependent kinase (AMPK)-regulated pathways was consistent with increased oxidative stress and with induction of apoptosis and reduction of preproinsulin gene transcription. CONCLUSIONS: The findings are consistent with impaired but partially compensated mechanisms of insulin secretion early in life, but progressive non-compensated impairments due to oxidative stress occurs by age 43 weeks.

Comparative cellular morphology suggesting the existence of resident dendritic cells within immune organs of salmonids.

This report is the first morphological description of cells that resemble dendritic cells, which appear to form resident populations within the spleen and anterior kidney of fish. Based on examination of three salmonid species, including, rainbow trout, brook trout, and Atlantic salmon, the cells were most abundant in the spleen, although they were always present in the anterior kidney. The cells appeared diffusely distributed, often near blood vessels of the spleen and kidney of healthy fish and within the epithelium, connective tissue, and blood vessels of rainbow trout gills with experimentally induced microsoporidial gill disease. The dendritic-like cells in this study contained granules that resemble Birbeck granules, which are considered to be morphological markers of Langerhans cells in mammals. The cells were approximately 6 mum in diameter and contained Birbeck-like (BL) granules localized near centrioles. Although the dendritic-like cells in the three salmonid species shared many similarities, morphological differences were found in the fine structure of the rod portion of the BL granules. Rainbow trout BL granules contained amorphous material, while the other salmonid species contained particulate material arranged in a square-lattice arrangement. The BL granules in the cells of Atlantic salmon had a narrow diameter and contained four layers of particulate material when sectioned longitudinally; two layers enveloped by the granule membrane and two central layers making up a central lamella, which is common in mammalian Birbeck granules.

Demonstration of infectious salmon anaemia virus (ISAV) endocytosis in erythrocytes of Atlantic salmon.

Infectious salmon anaemia (ISA) virus (ISAV) is a fish orthomyxovirus that has recently been assigned to the new genus Isavirus within the family Orthomyxoviridae. It possesses the major functional characteristics of the virus family including haemagglutinating, receptor destroying enzyme (RDE), and fusion activities associated with the virion surface proteins. It is generally accepted that ISAV agglutinates erythrocytes of several fish species and that the ISAV RDE activity dissolves this haemagglutination reaction except for Atlantic salmon (Salmo salar) erythrocytes. We used electron microscopy to examine the physical interaction between ISAV and erythrocytes from Atlantic salmon and rainbow trout (Oncorhynchus mykiss) during haemagglutination. We present evidence that ISAV enters into Atlantic salmon erythrocytes. Atlantic salmon erythrocytes incubated with ISAV for 4 hours showed endocytosis of the virus particles, which is consistent with virus infection. These observations suggest that the lack of dissolution of ISAV-induced haemagglutination of Atlantic salmon erythrocytes favours virus infection of the erythrocytes. Moreover, such a haemagglutination-infection phenotype is fundamentally different from haemagglutination by avian and mammalian orthomyxoviruses, and is indicative of a different pathogenesis for the fish orthomyxovirus.

Ultrastructural examination of the host inflammatory response within gills of netpen reared chinook salmon (Oncorhynchus tshawytscha) with Microsporidial Gill Disease.

The sequence of host changes following the rupture of spore-laden xenomas of the microsporidian Loma salmonae during Microsporidial Gill Disease of Salmon was deduced from ultrastructural examination of the gills of naturally infected, moribund, chinook salmon from a commercial aquaculture site. The gills contained many stages of parasite development suggesting fish were chronically exposed to the parasite. Intact xenomas were generally found beneath the endothelium in arteries and arterioles and were encapsulated by a layer of collagen containing fibroblasts sometimes joined by desmosomes. Xenoma dissolution was characterized by neutrophil infiltration and loss of the xenoma plasma membrane and encapsulation. The inflammatory responses associated with ruptured xenomas ranged from acute lesions, denoted by a marked neutrophil infiltration and vascular thrombosis, to chronic lesions with a macrophage-rich infiltrate variously accompanied by neovascularization and vascular remodelling. Dendritic-like cells and plasma cells were characteristic throughout. Basement membrane damage of the primary filament epithelium and subsequent transepithelial expulsion of spores were associated with severe inflammation. An unusual previously undescribed multifocal change, in which epithelial cells invaded deeply beyond the normal boundaries of the basement membrane, affected areas of gill filament epithelium with basement membrane damage. Some neutrophils that contained L. salmonae spores, or spore polar tube, displayed morphological changes that included irregular cell shape, cytoplasmic darkening associated with an abundance of free ribosomes, lysis of neighbouring cells, and type II nuclear clefts. Fusion of apparently intact neutrophils occurred in other areas of the lesion, where close contacts between neighbouring cells were established and in some areas plasma membrane fusion occurred. Closely associated neutrophils with intact plasma membranes were observed to contain type II nuclear clefts, abundant granules and rough endoplasmic reticulum. Other neutrophils in the lesion displayed type I nuclear pockets, which is suspected to be an early stage of apoptosis.

Pathological effects caused by chronic treatment of rainbow trout with indomethacin.

The purpose of this study was to determine adequate dose ranges and to test for side effects associated with chronic treatment of fish with indomethacin. Rainbow trout Oncorhynchus mykiss were orally treated with indomethacin at various nominal concentrations: a negative control (0 mg/kg) and low (5 mg/kg), medium (10 mg/kg), and high doses (15 mg/kg) daily for 30 d. A dose-associated mortality was observed, as cumulative mortality was 3, 13, and 33%, respectively, in the three indomethacin dose groups. No lesions were observed grossly or with histopathology in the control and low-dose treatment groups. Gross lesions were observed in the medium- and high-dose groups, including skin ulcers, abdominal distension, and necrosis of abdominal wall muscle. Histopathology of fish in the medium- and high-dosage groups revealed severe granulomatous peritonitis in which a large number of foreign body type giant cells were present around proteinaceous and plant material. The inflammatory response spread from the peritoneum through the somatic muscle to the epidermis, causing lesions within all layers of the skin. A large number of bacteria were noted within the peritonitis, observed both intracellularly and in large aggregates extracellularly. Perforations occurred within the anterior intestine, and the thick muscularis layer was replaced with inflammatory tissue. The present investigation shows that chronic indomethacin treatment produces gastrointestinal side effects in rainbow trout similar to those seen in mammals.

In vitro heterogeneity of osteogenic cell populations at various equine skeletal sites.

Bone cell cultures were evaluated to determine if osteogenic cell populations at different skeletal sites in the horse are heterogeneous. Osteogenic cells were isolated from cortical and cancellous bone in vitro by an explant culture method. Subcultured cells were induced to differentiate into bone-forming osteoblasts. The osteoblast phenotype was confirmed by immunohistochemical testing for osteocalcin and substantiated by positive staining of cells for alkaline phosphatase and the matrix materials collagen and glycosaminoglycans. Bone nodules were stained by the von Kossa method and counted. The numbers of nodules produced from osteogenic cells harvested from different skeletal sites were compared with the use of a mixed linear model. On average, cortical bone sites yielded significantly greater numbers of nodules than did cancellous bone sites. Between cortical bone sites, there was no significant difference in nodule numbers. Among cancellous sites, the radial cancellous bone yielded significantly more nodules than did the tibial cancellous bone. Among appendicular skeletal sites, tibial metaphyseal bone yielded significantly fewer nodules than did all other long bone sites. This study detected evidence of heterogeneity of equine osteogenic cell populations at various skeletal sites. Further characterization of the dissimilarities is warranted to determine the potential role heterogeneity plays in differential rates of fracture healing between skeletal sites.

The mechanical function and structure of aortic microfibrils in the lobster Homarus americanus.

Marfan syndrome, a connective tissue disorder affecting the cardiovascular system, is caused by mutations of fibrillin-based microfibrils. These mutations often affect the calcium-binding domains, resulting in structural changes to the proteins. It is hypothesized that these Ca+2 binding sites regulate the structure and mechanical properties of the microfibrils. The mechanical properties of fresh and extracted lobster aortic rings in calcium solutions (1, 13 and 30 mM Ca+2) were measured. Samples underwent amino acid compositional analysis. Antibodies were produced against the material comprising extracted aortic rings. The ultrastructure of strained and unstrained samples was examined using transmission electron microscopy. Calcium level altered the tangent modulus of fresh vessels. These rings were significantly stiffer when tested at 30 mM Ca+2 compared to rings tested at 1 mM Ca+2. Amino acid comparisons between extracted samples, porcine and human fibrillin showed compositional similarity. Immunohistochemical analysis showed that antibodies produced against the material in extracted samples localized to the known microfibrillar elements in the lobster aorta and cross-reacted with fibrillin microfibrils of mammalian ciliary zonules. Ultrastructurally, vessels incubated in low calcium solutions showed diffuse interbead regions while those incubated in physiological or high calcium solutions showed interbead regions with more defined lateral edges.